WO2015162100A1 - Adjuvants de vaccins - Google Patents

Adjuvants de vaccins Download PDF

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Publication number
WO2015162100A1
WO2015162100A1 PCT/EP2015/058535 EP2015058535W WO2015162100A1 WO 2015162100 A1 WO2015162100 A1 WO 2015162100A1 EP 2015058535 W EP2015058535 W EP 2015058535W WO 2015162100 A1 WO2015162100 A1 WO 2015162100A1
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seq
immunogen
lysm
antigen
adjuvant
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PCT/EP2015/058535
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English (en)
Inventor
Emrah ALTINDIS
Ugo D'ORO
Sabrina Liberatori
Elisabetta SOLDAINI
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Novartis Ag
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides

Definitions

  • This invention is in the field of vaccine adjuvants and immunogenic compositions. BACKGROUND OF THE INVENTION
  • Adjuvants are included in many current vaccines to enhance the immune response to antigens in the vaccines.
  • Aluminium salts such as aluminium hydroxide or aluminium phosphate, are the most common adjuvants and have been used for many years. More recently, oil-in-water adjuvants have been approved for use in vaccines, e.g. MF59TM, a squalene-in- water emulsion.
  • TLRs are Pattern Recognition Receptors (PRR) that recognise specific pathogen-associated molecular patterns (PAMPs) and stimulate the innate immune system.
  • PRR Pattern Recognition Receptors
  • a number of TLR agonists with adjuvant properties are known.
  • MPL 3-desacyl-4'- monophosphoryl lipid A
  • the Flagellin protein is a TLR5 agonist
  • IC31TM a mixture of immunostimulatory oligonucleotide and polycationic polymer
  • the LysM domain a well-known domain of about 40 amino acid residues found in a variety of enzymes involved in bacterial wall degradation [1] and thought to have a peptidoglycan binding function
  • the LysM domain is a TLR2 agonist.
  • the well- characterised LysM domain has been shown to function as a TLR agonist, in particular as a TLR2 agonist.
  • the LysM domain has been shown to act as an adjuvant: compositions containing LysM domains mixed with or fused to antigens are able to elicit robust immune responses against those antigens
  • LysM domains have been identified as useful for stimulating the innate immune system through TLRs, in particular TLR2.
  • LysM domains have been identified as able to increase the immunogenicity of immunogens that are co-administered with or joined to the LysM domains, and able to increase the titres of functional antibodies specific for the immunogens. Therefore, the enhanced immune response elicited by the LysM immunological adjuvants of the invention is not a generic pro-inflammatory response, but is a specific response that is useful for adjuvanting vaccine compositions.
  • the present invention provides an immunological adjuvant which is a TLR agonist comprising or consisting of a LysM domain, or comprising or consisting of a fragment of a LysM domain.
  • the invention also provides an immunological adjuvant that is a mimetic that mimics the adjuvant effects of a LysM domain or a fragment of the LysM domain.
  • the immunological adjuvants may be peptide adjuvants.
  • the invention also provides immunological adjuvants comprising or consisting of a nucleic acid molecule encoding these peptide adjuvants.
  • the immunological adjuvants of the invention are TLR agonists.
  • the immunological adjuvants of the invention are TLR2 agonists.
  • the invention also provides an immunogenic composition comprising (i) the immunological adjuvant of the invention and (ii) an immunogen.
  • immunogenic compositions may include one or more further immunogens and/or one or more further adjuvants.
  • the present invention provides an immunogenic composition
  • a polypeptide or peptide mimetic comprising or consisting of i) a LysM domain having TLR agonist activity; ii) a fragment of a LysM domain having at least 10 or 15 amino acids and including a tripeptide motif with the following formula: G/E/N/D - D/E/N - T/S/N; or iii) a mimetic of (i) or (ii) having TLR agonist activity; and (b) an immunogen.
  • the immunological adjuvant or immunogenic composition does not comprise gram-positive bacteria or particles derived from gram-positive bacteria to which the polypeptide or peptide mimetic is bound.
  • the polypeptide or peptide mimetic of the invention is not bound to gram-positive bacteria or particles derived from gram- positive bacteria
  • the immunogen(s) may be a protein or a polysaccharide.
  • the immunogen(s) may be fused to the immunological adjuvant or adjuvants of the invention.
  • the invention provides hybrid polypeptides comprising an adjuvant of the invention and an immunogen, as well as a nucleic acid molecule encoding such a hybrid polypeptide, and immunogenic compositions comprising this hybrid polypeptide.
  • the immunogen may be mixed with the immunological adjuvant of the invention.
  • the immunogen and the immunological adjuvant of the invention may therefore be separate molecules.
  • the invention also provides a process for preparing an immunogenic composition comprising a step of mixing (i) an immunological adjuvant of the invention and (ii) an immunogen.
  • the present invention further provides the use as an adjuvant of a polypeptide or peptide mimetic comprising or consisting of i) a LysM domain having TLR agonist activity; ii) a fragment of a LysM domain having at least 10 or 15 amino acids and including a tripeptide motif with the following formula: G/E/N/D - D/E/N - T/S/N; or iii) a mimetic of (i) or (ii) having TLR agonist activity.
  • the TLR agonist activity is TLR2 agonist activity.
  • the invention also provides a kit comprising: (i) a first container that contains an immunological adjuvant of the invention; and (ii) a second container that contains an immunogen.
  • a kit comprising: (i) a first container that contains an immunological adjuvant of the invention; and (ii) a second container that contains an immunogen.
  • the contents of the two containers can be combined (e.g. at the point of use) to form an immunogenic composition of the invention.
  • kits may include a third container that contains a further immunogen and/or a further adjuvant.
  • the invention also provides methods of treatment and medical uses involving the immunogenic compositions of the invention.
  • the invention provides a method of enhancing a protective immune response against an immunogen comprising administering an immunological adjuvant, polypeptide or peptide mimetic of the invention in combination with the immunogen.
  • the present invention also provides a method of inducing an immune response in a subject against an immunogen which method comprises administering to the subject an immunogenic composition, immunological adjuvant or polypeptide or peptide mimetic of the invention.
  • the immunological adjuvant of the invention comprising or consisting of a LysM domain may be a peptide.
  • Peptides comprising a LysM domain for use as adjuvants according to the invention can readily be identified by the skilled person by searching publicly available databases.
  • the PFAM database entry for the LysM domain having accession number PF01476 (http://pfam.sanger.ac.uk/family/lysM), for example, identifies all proteins known to contain a LysM domain and the peptide adjuvant of the invention may comprise or consist of the LysM domain sequence from any one of these proteins.
  • the PFAM database entry also provides hidden Markov models (profile HMMs) which can be used to search databases for additional peptides comprising a LysM domain and to confirm that a particular peptide comprises a LysM domain.
  • the LysM domain of the immunological adjuvant of the invention will generally comprise a beta sheet -alpha helix-alpha helix-beta sheet ( ⁇ ) secondary structure that is known to be typical of the LysM domains [2].
  • a LysM domain comprising this secondary structure is typically about 40 to 45 amino acids long, for example about 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 amino acids long.
  • LysM domains are known to contain inserts of between 5 and 20 amino acids, particularly between the two alpha helices, and/or between the second alpha helix and the second beta-sheet of the ⁇ secondary structure [3].
  • the LysM domain may thus be between about 35 and about 80 amino acids long, for example about 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids long.
  • the peptide adjuvants of the invention that consist of a LysM domain may thus consist of between about 38 and about 80 amino acids, for example they may consist of about 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids, or 35-70, 40-65 or 45-60 amino acids.
  • the peptide adjuvants of the invention that comprise a LysM domain may be longer; for example, they may comprise the complete sequence of a protein known to contain a LysM domain or a fragment of such a protein.
  • the immunological adjuvant of the invention consists of a LysM domain or a fragment of a LysM domain and does not comprise a full intact protein.
  • the present invention allows the provision of shorter peptide adjuvants that are TLR agonists.
  • the immunological adjuvant consists of a LysM domain of 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids and, optionally, 1-10 additional amino acids.
  • the LysM domain of the immunological adjuvant of the invention may comprise a consensus sequence that is typical of a LysM domain.
  • the HMM logo for the consensus sequence of the LysM domain is available in the PFAM database entry for the LysM domain having accession number PF01476 (also shown in Figure 1).
  • the LysM domain may comprise one or more residues that are indicated by the PFAM database entry to be particularly conserved in LysM domains.
  • the LysM domain of the immunological adjuvant of the invention may thus comprise a glycine (G), a glutamate (E), an asparagine (N) or an aspartate (D) at a position corresponding to amino acid 6 in the consensus sequence shown in Figure 1 ; an aspartate (D), glutamate (E) or asparagine (N) at a position corresponding to amino acid 7 in the consensus sequence shown in Figure 1 ; and/or a threonine (T), serine (S) or asparagine (N) at a position corresponding to amino acid 8 in the consensus sequence shown in Figure 1.
  • the LysM domain of the immunological adjuvant of the invention may thus comprise the tripeptide motif G/E/N/D - D/E/N - T/S/N preferably at a position located between the first beta sheet and the first alpha helix of the ⁇ secondary structure of the LysM domain.
  • the LysM domain may comprise the tripeptide motif GDT (SEQ ID NO:8), GDS (SEQ ID NO:9), GET (SEQ ID NO: 10) or GES (SEQ ID NO: 11), between the first beta sheet and the first alpha helix of the ⁇ secondary structure of the LysM domain.
  • the LysM domain of the immunological adjuvant of the invention may also comprise additional amino acids that are shown to be conserved in the consensus sequence in Figure 1.
  • LysM domain may comprise one or more of the following amino acids: a valine at a position corresponding to amino acid 3 in the consensus sequence shown in Figure
  • LysM domains of the immunological adjuvant according to the invention include LysM domains that comprise or consist of the amino acid sequence presented in SEQ ID NO: 1 (the LysM domain from the Streptococcus agalactiae protein SAG0032/Sip2, also referred to herein as GBS322), SEQ ID NO:2 (the LysM domain from the Streptococcus pneumoniae protein spr0096) or SEQ ID NO: 3 (the third LysM domain from the Staphylococcus aureus protein SAOUHSC_00427, also referred to herein as Sta0069), which are shown in the examples to be TLR agonists.
  • SEQ ID NO: 1 the LysM domain from the Streptococcus agalactiae protein SAG0032/Sip2, also referred to herein as GBS322
  • SEQ ID NO:2 the LysM domain from the Streptococcus pneumoniae protein spr0096
  • LysM domains for use in the present invention are the LysM domains of SEQ ID NO: l (also referred to as LysMl) and SEQ ID NO:3 (also referred to as LysM3c). These domains are particularly effective immunological adjuvants, as shown in the Examples.
  • Staphylococcus aureus protein Sta0069 also comprises two further LysM domains, named LysM3a and LysM3b, that can be used in accordance with the invention, and which are located at residues 29-71 and 93-135 of the protein (presented in SEQ ID NO:33 and SEQ ID NO:34).
  • LysM domains of the immunological adjuvant according to the invention include LysM domains that comprise or consist of the amino acid sequences presented in SEQ ID NO:24 (from Protein A of Staphylococcus aureus, UniProtKB ref: P02976) and SEQ ID NO:25 and SEQ ID NO:26 (from NlpD of MenB, UniProtKB ref: Q9JYP9).
  • the LysM domain of the immunological adjuvant of the invention may also comprise or consist of an amino acid sequence having at least 40% identity to SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:33 or SEQ ID NO:34 (e.g. at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) that retains the ability to act as a TLR agonist, particularly as a TLR2 agonist.
  • Adjuvants displaying identity with LysM domains that comprise or consist of the amino acid sequences presented in SEQ ID NOS: l, 2 and 3 may retain the secondary structure and/or conserved amino acids typical of LysM domain structure and considered important for function as a TLR agonist discussed above.
  • the immunological adjuvant of the invention comprises more than one LysM domain, such as 3 LysM domains, as present in SEQ ID NO:36 (LysM3abc from S. aureus Sta0096) and SEQ ID NO:37 (fusion of LysM3abc and S. aureus StaOl l - tested in Example 3).
  • Preferred immunological adjuvants of the invention comprise or consist of an amino acid sequence having at least 40% identity to SEQ ID NO:36 (e.g.
  • the immunological adjuvant according to the invention comprises or consists of the sequence represented by X(2)-[VILATF]-[KQRVETASN]-X- [GENDKRSAT]-[DENSQ]-[TSNIVLAYF]-[LCVIFAM]-[WSYEAETKQ]-[ILVF]-X(10-32)- [LIFVMT]-X(3)-[NQSAVT]-X(9-15)-[LIVPA]-X(2)-[GNDEKR]-X(2)-[LIVMF]-X(3-10), or a fragment thereof.
  • the invention also provides immunological adjuvants comprising nucleic acid molecules encoding any of the (poly)peptide adjuvants comprising a LysM domain described above.
  • the immunological adjuvant may also comprise or consist of a fragment of a LysM domain providing that this fragment retains the ability to act as a TLR agonist, particularly as a TLR2 agonist.
  • Fragments will typically comprise or consist of the tripeptide motif discussed above that is present at amino acids 6-8 of the LysM consensus sequence in Figure 1 and that is thought to be important to the TLR agonist activity of the LysM domain.
  • the immunological adjuvant of the invention is a fragment of a LysM domain, it may thus comprise or consist of the tripeptide motif G/E/N/D - D/E/N - T/S/N.
  • the fragment may comprise or consist of the tripeptide motif GDT (SEQ ID NO:8), GDS (SEQ ID NO:9), GET (SEQ ID NO: 10) or GES (SEQ ID NO: 1 1).
  • this tripeptide motif is located between the first beta sheet and the first alpha helix of a LysM domain having a typical beta sheet - alpha helix - alpha helix - beta sheet ( ⁇ ) secondary structure.
  • the immunological adjuvant of the invention of the invention is a fragment of a LysM domain comprising the tripeptide motif discussed above, it may thus additionally comprise the first alpha helix and the first beta sheet present in the full-length LysM domain.
  • Any fragment of the LysM domain may be used provided that it acts as a TLR agonist, particularly as a TLR2 agonist.
  • Fragments of LysM domains used as immunological adjuvants of the invention may be between 3 and 45 amino acids long, for example at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 10, 20, 25, 30, 35, or 40 amino acids long. In one embodiment the fragments are at least 10 or 15 amino acids long.
  • the immunological adjuvant is a fragment of a LysM domain that consists of up to 3, 5, 10, 15, 25, 30, 35 or 40 amino acids of a LysM domain and, optionally, 1-10 additional amino acids. In a related embodiment, the fragment consists of fewer than 35, 30 or 25 amino acids of a LysM domain.
  • fragments of the LysM domain comprise or consist of the first beta sheet and alpha helix of the ⁇ secondary structure linked by the tripeptide motif discussed above, they will typically comprise or consist of around 15 to 45, 15 to 35 or 15 to 25 amino acids. Such fragments will typically comprise or consist of amino acids corresponding to amino acids 1 to 15 of the consensus sequence for the LysM domain shown in Figure 1.
  • the invention also provides immunological adjuvants comprising nucleic acid molecules encoding any of the peptide adjuvants comprising a LysM domain fragment described above.
  • the immunological adjuvant of the invention may also be a mimetic of a peptide comprising or consisting of a LysM domain or fragment thereof described above, providing that this mimetic retains the ability to act as a TLR agonist, particularly as a TLR2 agonist.
  • the mimetic may be a peptide.
  • the LysM domain fragments that act as TLR agonists comprise a beta sheet - tripeptide motif - alpha helix structure. It is therefore envisaged that peptides comprising or consisting of a beta sheet-alpha helix structure and/or comprising or consisting of the tripeptide motif discussed above will act as TLR agonists, irrespective of whether they are LysM domains or fragments of LysM domains.
  • the invention provides an immunological peptide adjuvant that comprises or consists of the tripeptide motif G/E/N/D - D/E/N - T/S/N.
  • the immunological peptide adjuvant may comprise or consist of the tripeptide motif GDT (SEQ ID NO:8), GDS (SEQ ID NO:9), GET (SEQ ID NO: 10) or GES (SEQ ID NO: 11).
  • the invention also provides an immunological peptide adjuvant that comprises a beta- sheet alpha helix ( ⁇ ) secondary structure.
  • the immunological peptide may further comprise a tripeptide motif described above between the beta-sheet and the alpha helix.
  • the secondary structure and sequence of proteins are readily searchable in publicly available databases.
  • the skilled person can thus identify peptides having a beta-sheet alpha- helix secondary structure, peptides comprising the tripeptide motif G/E/N/D - D/E/N - T/S/N, and peptides having a beta-sheet linked to an alpha-helix by the tripeptide motif G/E/N/D - D/E/N - T/S/N by searching such databases.
  • the ScanProsite tool provided by the Swiss Institute of Bioinformatics and available at http://prosite.expasy.org/scanprosite/ can be used to search the Uniprot database for sequences comprising the LysM loop consensus sequence.
  • the JPred tool provided by the University of Dundee and available at ttp://www.compbio. dundee.ac.uk/www-jpred/ can be used to predict the secondary structure.
  • NMB2091 protein from Neisseria meningitidis An example of a protein identified by standard database searches as containing a beta- sheet linked to an alpha-helix by the tripeptide motif GDT but does not containing a comprise a LysM domain is the NMB2091 protein from Neisseria meningitidis.
  • the invention includes an immunological peptide adjuvant that comprises or consists of the amino acid sequences presented in SEQ ID NO: 12 (the fragment of NMB2091 comprising a beta-sheet linked to an alpha helix by a GDT loop).
  • the immunological adjuvant of the invention may also comprise or consist of an amino acid sequence having at least 40% identity to SEQ ID NO: 12, (e.g.
  • TLR agonist at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity
  • Such adjuvants may retain the secondary structure and/or tripeptide motif considered important for function as a TLR agonist discussed above.
  • the invention also provides immunological adjuvants comprising nucleic acid molecules encoding any of the peptide mimetic adjuvants described above.
  • the invention also includes synthetic mimetics that mimic the beta-sheet-alpha helix structure and/or the tripeptide motif that are considered important for TLR agonist function. TLR agonist activity
  • immunological adjuvants of the invention described above including peptides comprising or consisting of LysM domains, peptides comprising or consisting of fragments of LysM domains, and mimetics of these peptide and fragments, all function as TLR agonists.
  • the immunological adjuvants of the invention are TLR2 agonists.
  • Agonist activity of the adjuvants of the invention against TLR2 can be determined by standard assays. Companies such as Imgenex, Invivogen supply cell lines which are stably co- transfected with human TLR genes and NFkB, plus suitable reporter genes, for measuring TLR activation pathways. They are designed for sensitive, broadworking range dynamics, and can be used for high-throughput screening. The examples presented herein describe the detection of TLR agonist activity using HEK293T cells transfected with human TLR genes and with luciferase as a reporter gene.
  • the adjuvants of the invention may induce at least a 10-fold increase in TLR activity in a cell transfected with a TLR and a reporter gene.
  • the adjuvants of the invention may induce at least a 50-fold, 100-fold, 250-fold, 500-fold or 1000-fold increase in TLR activity in a cell transfected with a TLR and a reporter gene.
  • the adjuvant may induce such an increase in TLR2 activity.
  • the adjuvants of the invention may increase TLR2 activity by binding directly to TLR2.
  • the ability of an adjuvant of the invention to bind TLR2 may be determined by standard binding assays known in the art, for example by ELISAs or by the yeast two-hybrid screen.
  • an adjuvant of the invention and an antigen in the same molecule may be able to promote a more efficacious antigen-specific immune response, as shown in the Examples.
  • the adjuvant of the invention acts as a TLR agonist by binding directly to the TLR, including the adjuvant and antigen in the same molecule will target the antigen to the TLR.
  • the invention therefore provides a fusion protein comprising an immunological adjuvant of the invention and an immunogen or immunogens. These fusion proteins are also referred to herein as hybrid polypeptides. It will be appreciated that the immunogen will be heterologous to the protein from which the LysM domain is derived (i.e.
  • the native protein containing a LysM domain may contain immunogenic regions but such a protein is not considered to be a fusion protein or hybrid protein within the context of the present invention).
  • the hybrid polypeptide may contain multiple copies of the adjuvant of the invention and/or multiple copies of the immunogen(s).
  • Hybrid polypeptides may be represented by the formula
  • X is an immunological adjuvant described above which may be a peptide comprising or consisting of LysM domain or fragment thereof or a peptide mimetic thereof;
  • L is an optional linker sequence
  • A is an optional N-terminal sequence
  • B is an optional C-terminal sequence
  • n 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more;
  • n 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more;
  • p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.
  • Useful linkers are GSGS (SEQ ID NO: 13), GSGGGG (SEQ ID NO: 14 or GSGSGGGG (SEQ ID NO: 15), with the Gly-Ser dipeptide being formed from a BamHl restriction site, thus aiding cloning and manipulation, and the (Gly) 4 tetrapeptide being a typical poly-glycine linker.
  • Other suitable linkers, particularly for use as the final L n are a Leu-Glu dipeptide or Gly-Ser.
  • Linkers will usually contain at least one glycine residue to facilitate structural flexibility e.g. a -L- moiety may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more glycine residues.
  • -A- is preferably an oligopeptide (e.g. with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) which provides a N-terminus methionine e.g. Met-Ala-Ser, or a single Met residue.
  • the -A- moiety can provide the polypeptide's N-terminal methionine (formyl-methionine, fMet, in bacteria).
  • One or more amino acids may be cleaved from the N-terminus of a nascent -A- moiety, however, such that the -A- moiety in a mature polypeptide of the invention does not necessarily include a N-terminal methionine.
  • C-terminal amino acid sequences will be apparent to those skilled in the art, such as a glutathione-S-transferase, thioredoxin, 14kDa fragment of S. aureus protein A, a biotinylated peptide, a maltose-binding protein, an enterokinase flag, etc.
  • hybrid polypeptides include polypeptides that comprise or consist of the amino acid sequences presented in SEQ ID NO: 16 (the LysM domain from the Streptococcus agalactiae protein SAG0032/Sip2, also referred to herein as GBS322, fused to the Neisseria meningitidis P10 antigen), or SEQ ID NO: 17 (the LysM domain from the Streptococcus pneumoniae protein spr0096 fused to the Neisseria meningitidis P10 antigen).
  • SEQ ID NO: 16 the LysM domain from the Streptococcus agalactiae protein SAG0032/Sip2, also referred to herein as GBS322, fused to the Neisseria meningitidis P10 antigen
  • SEQ ID NO: 17 the LysM domain from the Streptococcus pneumoniae protein spr0096 fused to the Neisseria meningitidis P10 antigen
  • hybrid polypeptides according to the invention include the sequences presented in SEQ ID NO: 35 (the LysM domain from the Streptococcus agalactiae protein SAG0032/Sip2, also referred to herein as GBS322, fused to the Staphylococcus aureus StaOl l antigen) and SEQ ID NO: 37 (the three LysMs of Sta0069, named LysM3abc - SEQ ID NO: 36, fused to Staphylococcus aureus StaOl l antigen), and homologues thereof.
  • SEQ ID NO: 35 the LysM domain from the Streptococcus agalactiae protein SAG0032/Sip2, also referred to herein as GBS322, fused to the Staphylococcus aureus StaOl l antigen
  • SEQ ID NO: 37 the three LysMs of Sta0069, named LysM3abc - SEQ ID NO:
  • the hybrid polypeptide may also comprise or consist of an amino acid sequence having at least 70% identity to SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:37 (e.g. at least 75% 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) that retains the ability to act as a TLR agonist, particularly as a TLR2 agonist.
  • the invention also provides nucleic acid which encodes a hybrid polypeptide of the invention.
  • the immunological adjuvant of the invention can be administered to animals in combination with immunogens to induce an immune response.
  • the invention can be used with a wide range of immunogens, for treating or protecting against a wide range of diseases.
  • the immunogen may elicit an immune response that protects against a viral disease (e.g. due to an enveloped or non-enveloped virus), a bacterial disease (e.g. due to a Gram negative or a Gram positive bacterium), a fungal disease, a parasitic disease, an auto-immune disease, or any other disease.
  • the immunogen may also be useful in immunotherapy e.g. for treating a tumour/cancer, Alzheimer's disease, or an addiction.
  • the immunogen may take various forms e.g. a whole organism, an outer-membrane vesicle, a protein, a saccharide, a liposaccharide, a conjugate (e.g. of a carrier and a hapten, or of a carrier and a saccharide or liposaccharide), etc.
  • the immunogen is a protein.
  • the Examples demonstrate that the LysM domain adjuvants of the invention are particularly effective at enhancing the immune response elicited by protein immunogens.
  • the LysM domain adjuvants of the invention are effective at specifically enhancing the immune response, in particular specific antibody titres, against immunogens co-administered with or fused to the LysM domain.
  • This effect is observed for numerous different immunogens, and is not reliant on any sequence similarity between the LysM domain adjuvant and any immunogen, and does not involve cross-reactivity between the LysM domain adjuvant and any immunogen. Therefore, in certain embodiments wherein the immunogen is a protein, the sequence identity between the LysM domain and the immunogen is less than 35%, preferably less than 30%, 25%, 20%, 15% or 10%. Sequence similarity may be less than 60%, preferably less than 50%, 40% or 30%.
  • the immunogen may elicit an immune response against an influenza virus, including influenza A and B viruses.
  • influenza virus immunogen Various forms of influenza virus immunogen are currently available, typically based either on live virus or on inactivated virus.
  • Inactivated vaccines may be based on whole virions, split virions, or on purified surface antigens.
  • Influenza antigens can also be presented in the form of virosomes.
  • Hemagglutinin is the main immunogen in current inactivated vaccines, and vaccine doses are standardised by reference to HA levels, typically measured by SRID.
  • Existing vaccines typically contain about 15 ⁇ g of HA per strain, although lower doses can be used e.g. for children, or in pandemic situations, or when using an adjuvant.
  • compositions may include between 0.1 and 150 ⁇ g of HA per influenza strain, preferably between 0.1 and 50 ⁇ g e.g. 0.1-20 ⁇ g, 0.1-15 ⁇ g, 0.1-10 ⁇ g, 0.1-7 ⁇ g, 0.5 ⁇ g, etc.
  • Particular doses include e.g. about 45, about 30, about 15, about 10, about 7.5, about 5, about 3.8, about 3.75, about 1.9, about 1.5, etc. per strain. It is usual to include substantially the same mass of HA for each strain included in the vaccine e.g.
  • HA mass for each strain is within 10% of the mean HA mass per strain, and preferably within 5% of the mean.
  • dosing is measured by median tissue culture infectious dose (TCID 50 ) rather than HA content, and a TCID50 of between 10 6 and 10 8 (preferably between 10 6'5 -10 7'5 ) per strain is typical.
  • TCID 50 median tissue culture infectious dose
  • cell lines that support influenza virus replication may be used. The cell line will typically be of mammalian origin e.g. MDCK.
  • Influenza A virus immunogens may be from any suitable HA subtype strain e.g.
  • the immunogen may elicit an immune response against a Candida fungus such as C. albicans.
  • the immunogen may be a ⁇ -glucan, which may be conjugated to a carrier protein.
  • the carrier protein may be the LysM domain so that no additional carrier protein is required.
  • the glucan may include ⁇ -1,3 and/or ⁇ -1,6 linkages. Suitable immunogens include those disclosed in references 6 & 7.
  • the immunogen may elicit an immune response against a Streptococcus bacterium, including S.agalactiae, S.pneumoniae and S.pyogenes.
  • the immunogen may be a capsular saccharide, which may be conjugated to a carrier protein.
  • the carrier protein may be the LysM domain so that no additional carrier protein is required.
  • the saccharide may be from one or more of serotypes la, lb, II, III, and/or V.
  • S.pneumoniae the saccharide may be from one or more of serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and/or 23F.
  • polypeptide immunogens may be used to elicit a protective anti-streptococcal immune response e.g. comprising RrgB, as disclosed in reference 8.
  • the immunogen may elicit an immune response against a Staphylococcus bacterium, including S. aureus or S.epidermidis.
  • the immunogen may comprise an IsdA antigen, an IsdB antigen, a ClfA antigen, a ClfB antigen, a SdrD antigen, a Spa antigen, an EsxA antigen, an EsxB antigen, a Sta006 antigen, a hemolysin, and/or a StaOl l antigen.
  • Suitable S. aureus immunogens and their combinations are disclosed in reference 9. Preferred S.
  • aureus immunogens for use in the invention include Sta006, StaOl l, EsxA, EsxB and HLA.
  • a composition may comprise all 5 of these immunogens, and all 5 may be mixed with or fused to an immunological adjuvant of the invention.
  • EsxA may be used as a hybrid polypeptide with EsxB.
  • immunogens such as Sta006, StaO 1 1 and the EsxAB hybrid protein are altered by replacing, modifying or deleting the cysteine residues such that disulphide bond formation is eliminated, which prevents covalent dimer formation and improves antigen stability. Further preferred combinations of S.
  • aureus immunogens for use in the invention include at least two antigens selected from a HLA antigen, a sta006 antigen, a lukE antigen and a spa antigen, and optionally one or more of a StaOl l antigen, a EsxAB hybrid antigen and a clfA antigen.
  • an immunogen for use in the invention comprises or consists of a peptide sequence encoded by a sequence selected from the group consisting of: SEQ ID NO: 27 (S. aureus EsxAB), SEQ ID NO: 28 (S. aureus HLA H35L), SEQ ID NO: 29 (S. aureus LukE), SEQ ID NO: 30 (S.
  • SEQ ID NO: 31 S. aureus Sta006
  • SEQ ID NO: 32 S. aureus StaOl l - polypeptide sequence provided in SEQ ID NO: 38
  • homologues and fragments thereof SEQ ID NO: 38
  • the immunogen may elicit an immune response against a meningococcal bacterium (Neisseria meningitidis).
  • the immunogen may be a capsular saccharide, which may be conjugated to a carrier protein.
  • the carrier protein may be the immunological adjuvant of the invention comprising a LysM domain, a fragment of a LysM domain or a mimetic described above, so that no additional carrier protein is required.
  • Capsular saccharides are particularly useful for protecting against meningococcal serogroups A, C, W135 and/or Y.
  • polypeptide immunogens and/or outer membrane vesicles may be used to elicit a protective anti- meningococcal immune response, particularly for use against serogroup B e.g. as disclosed in reference 10.
  • a polypeptide immunogen that elicits an immune response against Neisseria meningitidis is the PI OA antigen comprising the amino acid sequence in SEQ ID NO: 18 or the same sequence without the histidine tag shown in SEQ ID NO: 19. For therapeutic use in humans, the sequence without the tag is preferred.
  • the immunogen may elicit an immune response against a hepatitis virus, such as a hepatitis A virus, a hepatitis B virus and/or a hepatitis C virus.
  • a hepatitis virus such as a hepatitis A virus, a hepatitis B virus and/or a hepatitis C virus.
  • the immunogen may be hepatitis B virus surface antigen (HBsAg). In some embodiments, though, the immunogen is not HBsAg (cf. ref. 1 1).
  • the immunogen may elicit an immune response against a respiratory syncytial virus.
  • Immunogens may be from a group A RSV and/or a group B RSV.
  • Suitable immunogens may comprise the F and/or G glycoproteins or fragments thereof e.g. as disclosed in references 12 & 13.
  • the immunogen may elicit an immune response against a Chlamydia bacterium, including C. trachomatis and C.pneumoniae.
  • Suitable immunogens include those disclosed in references 14, 15, 16, 17, 18, 19, 20.
  • the immunogen may elicit an immune response against an Escherichia coli bacterium, including extraintestinal pathogenic strains. Suitable immunogens include those disclosed in references 21, 22, 23, 24. [68] The immunogen may elicit an immune response against a coronavirus, such as the human SARS coronavirus. Suitable immunogens may comprise the spike glycoprotein.
  • the immunogen may elicit an immune response against a Helicobacter pylori bacterium.
  • Suitable immunogens include CagA [25, 26, 27, 28], VacA [29,30], and/or NAP [31, 32, 33].
  • the immunogen may elicit an immune response against rabies virus.
  • a suitable immunogen is an inactivated rabies virus [34, RabAvertTM].
  • the immunogen may elicit an immune response against a human papillomavirus.
  • Useful immunogens are LI capsid proteins, which can assemble to form structures known as virus-like particles (VLPs).
  • the VLPs can be produced by recombinant expression of LI in yeast cells (e.g. in S.cerevisiae) or in insect cells (e.g. in Spodoptera cells, such as S.frugiperda, or in Drosophila cells).
  • yeast cells plasmid vectors can carry the LI gene(s); for insect cells, baculovirus vectors can carry the LI gene(s).
  • the composition includes LI VLPs from both HPV-16 and HPV-18 strains. This bivalent combination has been shown to be highly effective [35]. In addition to HPV-16 and HPV-18 strains, it is also possible to include LI VLPs from HPV-6 and HPV-11 strains.
  • the immunogen may elicit an immune response against a tumour antigen, such as MAGE-1, MAGE-2, MAGE-3 (MAGE-A3), MART-l/Melan A, tyrosinase, gplOO, TRP-2, etc.
  • a tumour antigen such as MAGE-1, MAGE-2, MAGE-3 (MAGE-A3), MART-l/Melan A, tyrosinase, gplOO, TRP-2, etc.
  • the immunogen may elicit an immunotherapeutic response against lung cancer, melanoma, breast cancer, prostate cancer, etc.
  • the immunogen may elicit an immune response against a hapten conjugated to a carrier protein, where the hapten is a drug of abuse [36].
  • hapten conjugated to a carrier protein
  • examples include, but are not limited to, opiates, marijuana, amphetamines, cocaine, barbituates, glutethimide, methyprylon, chloral hydrate, methaqualone, benzodiazepines, LSD, nicotine, anticholinergic drugs, antipsychotic drugs, tryptamine, other psychomimetic drugs, sedatives, phencyclidine, psilocybine, volatile nitrite, and other drugs inducing physical and/or psychological dependence.
  • the immunogenic compositions and fusion proteins may contain more than one immunogen, for example they may contain 2, 3, or 4 different immunogens. Additional adjuvants
  • Immunogenic compositions of the invention may also comprise one or more additional adjuvants.
  • Additional adjuvants which may be used in compositions of the invention include, but are not limited to mineral salts, such as aluminium salts and calcium salts.
  • the invention includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates, etc. [e.g. see chapters 8 & 9 of ref. 37], or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption being preferred.
  • the mineral containing compositions may also be formulated as a particle of metal salt.
  • Aluminium salts are particularly preferred adjuvants for use in combination with the LysM immunological adjuvant of the invention.
  • the Examples demonstrate that LysM domains are particularly effective for enhancing the immune response to immune compositions comprising alum.
  • aluminium hydroxide typically aluminium oxyhydroxide salts, which are usually at least partially crystalline.
  • Aluminium oxyhydroxide which can be represented by the formula AIO(OH)
  • IR infrared
  • the degree of crystallinity of an aluminium hydroxide adjuvant is reflected by the width of the diffraction band at half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes.
  • aluminium hydroxide adjuvants The surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption.
  • a fibrous morphology e.g. as seen in transmission electron micrographs
  • the pi of aluminium hydroxide adjuvants is typically about 11 i.e. the adjuvant itself has a positive surface charge at physiological pH.
  • Adsorptive capacities of between 1.8-2.6 mg protein per mg Al +++ at pH 7.4 have been reported for aluminium hydroxide adjuvants.
  • the adjuvants known as "aluminium phosphate” are typically aluminium hydroxyphosphates, often also containing a small amount of sulfate (i.e. aluminium hydroxyphosphate sulfate). They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt. Hydroxyphosphates generally have a PO 4 /AI molar ratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished from strict AIPO 4 by the presence of hydroxyl groups. For example, an IR spectrum band at 3164cm "1 (e.g. when heated to 200°C) indicates the presence of structural hydroxyls [ch. 9 of ref. 37].
  • the P0 4 /A1 3+ molar ratio of an aluminium phosphate adjuvant will generally be between 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably 0.95 ⁇ 0.1.
  • the aluminium phosphate will generally be amorphous, particularly for hydroxyphosphate salts.
  • a typical adjuvant is amorphous aluminium hydroxyphosphate with PO 4 /AI molar ratio between 0.84 and 0.92, included at 0.6mg Al 3+ /ml.
  • the aluminium phosphate will generally be particulate (e.g. plate-like morphology as seen in transmission electron micrographs). Typical diameters of the particles are in the range 0.5-20 ⁇ (e.g. about 5-10 ⁇ ) after any antigen adsorption.
  • Adsorptive capacities of between 0.7-1.5 mg protein per mg Al +++ at pH 7.4 have been reported for aluminium phosphate adjuvants.
  • Suspensions of aluminium salts used to prepare compositions of the invention may contain a buffer (e.g. a phosphate or a histidine or a Tris buffer), but this is not always necessary.
  • the suspensions are preferably sterile and pyrogen-free.
  • a suspension may include free aqueous phosphate ions e.g. present at a concentration between 1.0 and 20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM.
  • the suspensions may also comprise sodium chloride.
  • an adjuvant component includes a mixture of both an aluminium hydroxide and an aluminium phosphate. In this case there may be more aluminium phosphate than hydroxide e.g. a weight ratio of at least 2: 1 e.g. >5: 1, >6: 1, >7: 1, >8: 1, >9: 1, etc.
  • the concentration of Al +++ in a composition for administration to a patient is preferably less than lOmg/ml e.g. ⁇ 5 mg/ml, ⁇ 4 mg/ml, ⁇ 3 mg/ml, ⁇ 2 mg/ml, ⁇ 1 mg/ml, etc.
  • a preferred range is between 0.3 and lmg/ml.
  • a maximum of ⁇ 0.85mg/dose is preferred.
  • compositions of the invention include a mixture of immunostimulatory oligonucleotide and polycationic polymer known as IC31TM [38, 39, 40], which is an adsorptive complex of oligodeoxynucleotide ICICICICICICICICICICIC (SEQ ID NO: 20) and polycationic oligopeptide KLKLLLLLKLK (SEQ ID NO:21)
  • the oligonucleotide and oligopeptide can be mixed together at various ratios, but they will generally be mixed with the peptide at a molar excess.
  • the molar excess may be at least 5: 1 e.g. 10: 1, 15: 1, 20: 1, 25: 1, 30; 1, 35: 1, 40: 1 etc.
  • a molar ratio of about 25: 1 is ideal [41,42]. Mixing at this excess ratio can result in formation of insoluble particulate complexes between oligonucleotide and oligopeptide.
  • the oligonucleotide and oligopeptide will typically be mixed under aqueous conditions e.g. a solution of the oligonucleotide can be mixed with a solution of the oligopeptide with a desired ratio.
  • the two solutions may be prepared by dissolving dried (e.g. lyophilised) materials in water or buffer to form stock solutions that can then be mixed.
  • the complexes can be maintained in aqueous suspension e.g. in water or in buffer.
  • Typical buffers for use with the complexes are phosphate buffers (e.g. phosphate-buffered saline), Tris buffers, Tris/sorbitol buffers, borate buffers, succinate buffers, citrate buffers, histidine buffers, etc.
  • phosphate buffers e.g. phosphate-buffered saline
  • Tris buffers Tris buffers
  • Tris/sorbitol buffers borate buffers
  • succinate buffers citrate buffers
  • histidine buffers etc.
  • complexes may sometimes be lyophilised.
  • Complexes in aqueous suspension can be centrifuged to separate them from bulk medium (e.g. by aspiration, decanting, etc.). These complexes can then be re-suspended in an alternative medium if desired.
  • the immunogenic compositions of the invention comprise aluminium salts and/or IC31TM as additional adjuvants. In some embodiments, the immunogenic compositions of the invention comprise an aluminium salt and a TLR7 ligand.
  • the invention provides pharmaceutical compositions comprising a LysM domain immunological adjuvant of the invention and an immunogen, such as an immunogen as defined above.
  • the immunological adjuvant of the invention and the immunogen may part of the same fusion protein, they may be fused or conjugated together, or the immunological adjuvant of the invention and the immunogen may be separate molecules that are mixed together in the pharmaceutical composition of the invention.
  • LysM immunological adjuvants are particularly effective at enhancing the immune response against a particular immunogen when administered together with the immunogen in a composition.
  • Immunological compositions of the invention usually include components in addition to the immunological adjuvant describe above, e.g. they typically include one or more pharmaceutically acceptable component. Such components may also be present in immunogenic compositions of the invention, originating either in the adjuvant composition or in another composition. A thorough discussion of such components is available in reference 45.
  • a composition may include a preservative such as thiomersal or 2-phenoxyethanol. It is preferred that the vaccine should be substantially free from (e.g. ⁇ lC ⁇ g/ml) mercurial material e.g. thiomersal-free. Vaccines containing no mercury are more preferred. Preservative- free vaccines are particularly preferred, a-tocopherol succinate can be included as an alternative to mercurial compounds in influenza vaccines.
  • a preservative such as thiomersal or 2-phenoxyethanol. It is preferred that the vaccine should be substantially free from (e.g. ⁇ lC ⁇ g/ml) mercurial material e.g. thiomersal-free. Vaccines containing no mercury are more preferred. Preservative- free vaccines are particularly preferred, a-tocopherol succinate can be included as an alternative to mercurial compounds in influenza vaccines.
  • a composition may include a physiological salt, such as a sodium salt.
  • a physiological salt such as a sodium salt.
  • Sodium chloride ( aCl) is preferred, which may be present at between 1 and 20 mg/ml.
  • Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and/or magnesium chloride, etc.
  • Compositions may have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, maybe within the range of 280-330 mOsm/mg or 290-310 mOsm/kg.
  • the pH of a composition will generally be between 5.0 and 8.1, and more typically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8.
  • a composition is preferably sterile.
  • a composition is preferably non-pyrogenic e.g. containing ⁇ 1 EU (endotoxin unit, a standard measure) per dose, and preferably ⁇ 0.1 EU per dose.
  • a composition is preferably gluten free.
  • a useful composition comprises a histidine buffer (e.g. 10 mM histidine buffer), sodium chloride (e.g. 9mg/ml sodium chloride) and an aluminium hydroxide adjuvant (e.g. 2 mg/ml Al +++ ).
  • a histidine buffer e.g. 10 mM histidine buffer
  • sodium chloride e.g. 9mg/ml sodium chloride
  • an aluminium hydroxide adjuvant e.g. 2 mg/ml Al +++
  • An immunogenic composition may include material for a single immunisation, or may include material for multiple immunisations (i.e. a 'multidose' kit).
  • a preservative is useful in multidose arrangements.
  • the compositions may be contained in a container having an aseptic adaptor for removal of material.
  • compositions will generally be in aqueous form at the point of administration.
  • Vaccines are typically administered in a dosage volume of about 0.5 ml, although a half dose (i.e. about 0.25 ml) may sometimes be administered e.g. to children.
  • a composition may be administered in a higher dose e.g. about 1 ml e.g. after mixing two 0.5 ml volumes.
  • Suitable containers for the immunological adjuvants, immunogenic compositions and kit components of the invention include vials, syringes (e.g. disposable syringes), etc. These containers should be sterile. The containers can be packaged together to form a kit e.g. in the same box.
  • the vial can be made of a glass or plastic material.
  • the vial is preferably sterilized before the composition is added to it.
  • vials are preferably sealed with a latex-free stopper, and the absence of latex in all packaging material is preferred.
  • the vial may include a single dose of vaccine, or it may include more than one dose (a 'multidose' vial) e.g. 10 doses.
  • Useful vials are made of colorless glass. Borosilicate glasses are preferred to soda lime glasses. Vials may have stoppers made of butyl rubber.
  • a vial can have a cap (e.g. a Luer lock) adapted such that a syringe can be inserted into the cap.
  • a vial cap may be located inside a seal or cover, such that the seal or cover has to be removed before the cap can be accessed.
  • a vial may have a cap that permits aseptic removal of its contents, particularly for multidose vials.
  • the syringe may have a needle attached to it. If a needle is not attached, a separate needle may be supplied with the syringe for assembly and use. Such a needle may be sheathed.
  • the plunger in a syringe may have a stopper to prevent the plunger from being accidentally removed during aspiration.
  • the syringe may have a latex rubber cap and/or plunger.
  • Disposable syringes contain a single dose of vaccine.
  • the syringe will generally have a tip cap to seal the tip prior to attachment of a needle, and the tip cap may be made of a butyl rubber. If the syringe and needle are packaged separately then the needle is preferably fitted with a butyl rubber shield.
  • Useful syringes are those marketed under the trade name "Tip-Lok"TM.
  • Containers may be marked to show a half-dose volume e.g. to facilitate delivery to children.
  • a syringe containing a 0.5ml dose may have a mark showing a 0.25ml volume.
  • an individual container may include overfill e.g. of 5-20% by volume.
  • compositions of the invention are suitable for administration to human subjects, and the invention provides a method of raising an immune response in a subject, comprising the step of administering an immunogenic composition of the invention to the subject.
  • the invention also provides a method of raising an immune response in a subject, comprising the step of mixing the contents of the containers of a kit of the invention and administering the mixed contents to the subject.
  • the invention also provides a composition or kit of the invention for use as a medicament e.g. for use in raising an immune response in a subject.
  • the invention also provides the use of an immunological adjuvant as described above, in the manufacture of a medicament for raising an immune response in a subject.
  • This medicament may be administered in combination with an immunogen such as the immunogens described above.
  • the invention also provides a method of enhancing the protective immune response elicited against an immunogen, comprising administering a LysM immunological adjuvant of the invention in combination with the immunogen.
  • the immunological adjuvant enhances the protective immune response against a specific immunogen that is joined to the immunological adjuvant or is administered in combination with the immunological adjuvant.
  • administering a LysM immunological adjuvant enhances the adaptive immune response against the immunogen.
  • administering a LysM immunological adjuvant enhances the innate immune response against the immunogen.
  • the invention provides a method of stimulating the innate immune system by activating TLRs (particularly TLR2) using an immunological adjuvant of the invention.
  • the invention provides a LysM immunological adjuvant for use in a method of stimulating dendritic cells, for example in order to elicit a protective immune response against an immunogen.
  • a LysM immunological adjuvant enhances the adaptive immune response against a specific antigen and activates the innate immune response at the same time.
  • the invention provides an immunological adjuvant comprising or consisting of a LysM domain from a first protein, or a fragment thereof, for use in a method of eliciting a protective immune response against a second, different protein, wherein the method comprises administering the immunological adjuvant in combination with the second protein.
  • the invention provides an immunological adjuvant comprising or consisting of a LysM domain from a first organism, or a fragment thereof, for use in a method of eliciting a protective immune response against a second, different organism, wherein the method comprises administering the immunological adjuvant in combination with an immunogen from the second organism.
  • the LysM immunological adjuvant of the invention increases antibody titres (measured, for example by ELISA or a bactericidal assay), such as the titre of neutralising antibodies, or increases cytokine production (measured, for example by ELISA, flow cytometry or PCR), e.g. by at least a factor of 1.5, 2, 4, 10, 50, 100, 500 or 1000, relative to the immune response elicited by the antigen in the absence of the LysM immunological adjuvant.
  • the LysM immunological adjuvant of the invention increases the protection provided by an immunogen.
  • the LysM immunological adjuvants of the invention are particularly effective for increasing the titre of antibodies specific for the coadministered immunogen. Therefore, in preferred embodiments, the invention provides a method for eliciting an increased titre of antibodies (preferably neutralising, protective antibodies) specifically reactive with an immunogen comprising co-administering or fusing the immunogen with a LysM immunological adjuvant. The invention also provides LysM immunological adjuvants for use in such methods.
  • the invention provides a LysM immunological adjuvant of the invention for use in a method of enhancing antibody titres against a specific immunogen that is fused to the immunological adjuvant or that is administered in combination with the immunological adjuvant.
  • compositions of the invention can be administered in various ways.
  • the usual immunisation route is by intramuscular injection (e.g. into the arm or leg), but other available routes include subcutaneous injection, intranasal, oral, buccal, sublingual, intradermal, transcutaneous, transdermal, etc.
  • Immunogenic compositions prepared according to the invention may be used as vaccines to treat both children and adults.
  • a subject may be less than 1 year old, 1-5 years old,
  • Subjects for receiving the vaccines may be elderly (e.g. >50 years old, >60 years old, and preferably >65 years), the young (e.g.
  • Aluminium salt adjuvants are routinely used in infant populations, and IC31TM has also been effective in this age group [46].
  • the vaccines are not suitable solely for these groups, however, and may be used more generally in a population.
  • Treatment can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Administration of more than one dose (typically two doses) is particularly useful in immunologically naive subjects. Multiple doses will typically be administered at least 1 week apart (e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 16 weeks, etc.).
  • GI numbering is used above.
  • a GI number, or “Genlnfo Identifier” is a series of digits assigned consecutively to each sequence record processed by NCBI when sequences are added to its databases. The GI number bears no resemblance to the accession number of the sequence record.
  • a sequence is updated (e.g. for correction, or to add more annotation or information) then it receives a new GI number. Thus the sequence associated with a given GI number is never changed.
  • composition comprising X may consist exclusively of X or may include something additional e.g. X + Y.
  • Antibodies will generally be specific for their target. Thus they will have a higher affinity for the target than for an irrelevant control protein, such as bovine serum albumin.
  • references to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. 55.
  • a preferred alignment is determined by the Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62.
  • the Smith- Waterman homology search algorithm is disclosed in ref. 56.
  • Figure 1 HMM logo for the consensus sequence of the LysM domain from the PFAM database, accession number PF01476.
  • FIG. 2 Testing of the LysM domains from the Streptococcus agalactiae protein GBS322 and the Staphylococcus aureus protein Sta0069 for TLR agonist activity.
  • the reverse LysMl sequence (SeqlRev, SEQ ID NO:4) was used as a negative control and the Pam 3 CSK 4 lipopeptide as a positive control.
  • the reverse LysMl sequence (SeqlRev) was used as a negative control and LPS as a positive control.
  • TLR2 agonists having the amino acid sequences NPPTT (Seq VI 54, SEQ ID NO:5), CGPKL (Seq VI 65, SEQ ID NO:6) and GGKLS (Seq VI 69, SEQ ID NO:7) were assessed for TLR agonist activity.
  • the peptides did not induce luciferase activity in HEK293T cells transfected with TLR2 (HEK293T-TLR2-Luc). The Pam 3 CSK 4 lipopeptide was used as a positive control.
  • B The peptides did not induce luciferase activity in HEK293T-TLR4-Luc cells transfected with TLR4. LPS was used as a positive control.
  • C The peptides did not induce luciferase activity in HEK293T-Luc cells without TLRs.
  • FIG. 4 Adjuvant activity of the LysM domain sequences from the Streptoccocus agalactiae protein GBS322 (LysMl) or the Streptococcus pneumoniae protein spr0096 (LysM2) fused to or mixed with the P 10A antigen from Neisseria meningitidis.
  • A ELISA titres of immune sera from mice with LysMl mixed with or fused to the P 10A antigen, with or without alum and IC31, against Neisseria meningitis strains following immunisation
  • B ELISA titres of immune sera from mice with LysM2 mixed with or fused to the P10A antigen, with or without alum and IC31, against Neisseria meningitis strains following immunisation.
  • FIG. 5 Adjuvant activity of the LysM domain sequences from the Streptoccocus agalactiae protein GBS322 (LysMl) or the Streptococcus pneumoniae protein spr0096 (LysM2) fused to or mixed with the PI OA antigen from Neisseria meningitidis.
  • BCA Percentage of bactericidal assay
  • FIG. 6 Adjuvant activity of the LysM domain sequences from the Streptoccocus agalactiae protein GBS322 (LysMl) and from the S. aureus protein Sta0069 (LysM3abc) fused to the StaOl l antigen from 5 * . aureus.
  • Specific IgG titres 13 days after immunisation with alum and with alum-TLR7 are reported.
  • LysM domain was identified as a new PAMP candidate. This hypothesis was tested using two strategies: 1) testing of synthetic LysM sequences thereof for adjuvant activity in cell-based assays; and 2) testing of fusion proteins comprising LysM sequences fused to immunogens for adjuvant activity.
  • Peptide sequences comprising i) the LysM domain of the protein SAG0032 from Streptococcus agalactiae, or ii) the LysM domain of the protein Sta069 from Staphylococcus aureus were synthesized and tested for TLR agonist activity in TLR-transfected HEK293 cells with a luciferase reporter system driven by an NF-kB promoter.
  • Peptide sequences comprising the LysM domain of the protein SAG0032 from Streptococcus agalactiae were also tested in for the ability to induce cytokine production in murine spleen cells from wild-type and TLR2 knock-out mice.
  • LysMlrev A peptide of the same length as the LysMl peptide containing the reverse LysMl sequence (LysMlrev) was also synthesized and used as a negative control. The sequence of this peptide was as follows. [135] Sequence of LysMlrev (Seqlrev or SeqlR):
  • HEK293T-Luc cells stably transfected with human TLR2 (HEK293T-TLR2-Luc), stably transfected with human TLR4 (HEK293T-TLR4-Luc) or without any TLR transfection (HEK293T-Luc) were cultured overnight at 5 x 10 4 cells/well in microclear 96-well plates (Greiner bio-one). Cells were stimulated with serial dilutions of the synthetic peptides in duplicate for 6 hours and then lysed with cell culture lysis reagent (Promega), according to manufacturer's instructions. Luciferase assay substrate (Promega) was added and luciferase activity measured by SpectraMax L microplate reader (Molecular Devices).
  • Luciferase activity was expressed as fold induction over non stimulated cells.
  • the synthetic peptides were resuspended at 20 ⁇ in DMSO and then diluted in culture medium.
  • the Pam 3 CSK 4 lipopeptide and LPS were used on HEK293T-TLR2-Luc and HEK293T-TLR4- Luc, respectively.
  • mice were sacrified and spleens were removed aseptically, the cells were isolated by use of a loosely fitting Dounce homogenizer, washed, adjusted to a concentration of 4 x 10 6 cells per ml, and grown in flat-bottom microwell plates (Nunc, Roskilde, Denmark), in RPMI
  • 1640 medium supplemented with glutamine, HEPES (N-2-hydroxyethylpiperazine- N'-2- ethanesulfonic acid), 5 x 10 "5 M 2-mercaptoethanol, antibiotics, and 10% heat-inactivated fetal calf serum.
  • the peptides were added in different amounts (100 ⁇ , 20 ⁇ , 4 ⁇ , 0.8 ⁇ , 0.16 ⁇ , 0.03 ⁇ , 0.006 ⁇ ) to cell suspension. Cells were incubated at 37°C in a humidified CO 2 incubator, and supernatants were harvested after 24 h. Supernatants from three different wells were pooled and stored frozen at -20°C until the assay was performed.
  • the LysMl peptide (Seql) and the LysM3c peptide (Seq3) comprising the LysM domain from GBS322 and Sta0069 respectively specifically activated HEK293T cells transfected with TLR2 (HEK293T-TLR2-Luc) to produce luciferase activity ( Figure 2A).
  • the LysMl and LysM3c peptides did not induce luciferase activity in HEK293T- TLR4-Luc cells transfected with TLR4 ( Figure 2B) or without TLRs ( Figure 2C).
  • the negative control LysMlrev peptide (Seqlrev) did not induce luciferase activity in any of the experiments.
  • LysMl peptide was also able to induce cytokine production in spleen cells from wild-type mice but this activation was lost when spleen cells from TLR2 knock-out mice were used (results not shown).
  • LysMl and LysM3c peptides are able to stimulate the innate immune system through a TLR2-mediated mechanism.
  • TLR2 agonists these peptides are likely to be useful as new intramolecular adjuvants.
  • LysM domains from two unrelated proteins both display TLR2 agonist activity indicates that TLR2 agonist activity is likely to be shared by LysM domains from other proteins.
  • LysM domain sequences fused to specific antigens were tested for their ability to promote protective immunity in a relevant animal model and to measure antibody titres/bactericidal activity of corresponding sera.
  • Purified recombinant fusion proteins made of a MenB antigen fused to the LysM domain from SAG0032 were tested in the animal model with and without Alum, and in the absence of supplemental TLR ligands. The comparison was then made with the same antigen fused to the LysM domain or supplemented with other TLR ligands.
  • Serum bactericidal activity against N. meningitidis strains was evaluated as follows. SBA against N. meningitidis strains was evaluated with pooled baby rabbit serum used as complement source. Briefly, N. meningitidis strains were grown overnight on chocolate agar plates at 37°C in 5% CO 2 . Colonies were inoculated in Mueller-Hinton broth, containing 0.25% glucose, to reach an OD 6 2o of 0.05-0.08 and incubated at 37°C with shaking until the OD620 reached the value of 0.23-0.24.
  • Bacteria were diluted in the assay buffer ⁇ at the working dilution of 10 4 CFU/mL. All sera to test were heat inactivated for 30 minutes at 56°C. The total volume in each well was 50 ⁇ 1 with 25 ⁇ 1 of serial two-fold dilutions of the test serum, 12.5 ⁇ 1 of bacteria at the working dilution and 12.5 ⁇ 1 of baby rabbit complement.
  • Negative controls included bacteria incubated, separately, with the complement serum without the test serum and with test immune sera and heat inactivated complement.
  • the negative controls were plated on Mueller-Hinton agar plates using the tilt method (time 0). The plate was incubated for 1 hour at 37°C, then each sample were spotted in duplicate on Mueller-Hinton agar plates and the controls were plated on Mueller-Hinton agar plates using the tilt method (time 60). The Mueller Hinthon agar plates and incubated for 18 hours at 37°C +5% CO 2 and the colonies were counted.
  • Serum bactericidal titres were defined as the serum dilution resulting in 50% decrease in colony forming units (CFU) per ml after 60 min incubation of bacteria in the reaction mixture, compared to control CFU per ml at time 0.
  • CFU colony forming units
  • Assay buffer for MenB strains Dulbecco's Phosphate Buffered Saline (DPBS), (SIGMA) + 1% (w/v) BSA + 0.1% glucose was used.
  • MenB antigens in three different variants, were immobilized on 96 well Greiner plates, washed with PBT as washing buffer and with PVP as saturation buffer. 100 ⁇ of diluted sera were added to each well, incubated for 2 hours at 37°C and then the antigen/antibody reaction was revealed using HRP-conjugated rabbit anti-mouse serum in presence of the substrate H2S04 and values were read at OD492-630.
  • the ELISA titres were calculated arbitrarily as the dilution of sera which gave an OD492-630 value of 0.4 above the level of preimmune sera. The ELISA was considered positive when the dilution of sera with OD492-630 of 0.4 was higher than 1 :400.
  • LysMl domain is particularly effective at enhancing the response to compositions that comprise alum and/or IC31. This effect is most pronounced when LysMl is fused to the antigen, in the presence of alum. These data also show that the response elicited using LysM domains is effective against a range of strains. These data suggest that recombinant protein vaccines could be developed that include in the same molecule the antigen and the intramolecular adjuvant in the form of a LysM domain as TLR2 ligand.
  • LysM fusion proteins The ability of LysM fusion proteins to increase specific antibody titres was demonstrated for further fusion proteins comprising the S. aureus StaOl l antigen fused to the LysMl domain (S. agalactiae GBS322) or to the 3 LysM domains of S. aureus Sta069, termed LysM3abc. These fusions were tested in alum-adjuvanted IP vaccine compositions and in alum-TLR7 compositions. Control compositions included the LysMl domain with alum, the Sta069 antigen with alum, and the StaO 11 antigen with alum.
  • compositions listed in Figure 6 were administered to groups of eight female CD1 mice at 5 weeks of age. Each mouse received IP immunisations of 200 ⁇ total volume, comprising 20 ⁇ g antigen dose and 2 mg/ml alum. The immunisations were administered on day 2 and blood samples were taken on days 1 and 15.
  • Antibody titers were measured by Luminex assay using protein-coupled microspheres.
  • MicroPlex microspheres (Luminex Corp), according to the manufacturer's instructions. To determine serum titers, the beads were incubated with mouse sera raised against the different recombinant constructs, washed twice PBS, and then incubated with phycoerythrin-conjugated secondary antibodies. IgG measurements were determined on the Luminex 200 analyzer using Bio-Plex Manager 5.0 software (Bio-Rad, Hercules, CA). Tests were performed in duplicate, and the mean fluorescence intensity (MFI) was determined. The limit of quantification (LOQ) of the assay was determined at an MFI of 100 and was considered the threshold for positive results.
  • MFI mean fluorescence intensity
  • Figure 6 provides the StaOl 1 -specific IgG titres measured on day 15, 13 days after immunisation.
  • the titre elicited by the LysM3abc-Sta01 1/alum composition was higher than the titre elicited by the StaOl 1/alum composition, although the difference was not statistically significant.
  • neither LysMl nor Sta069 alone in alum were able to elicit cross-reactive antibody titres against StaOl 1, indicating that the increased titre in the fusion is the actual result of an adjuvant effect of these molecules.
  • SAOUHSC 00427 also referred to herein as Sta0069.
  • SEQ ID NO:20 IC31 oligodeoxynucleotide.
  • SEQ ID NO: 27 S. aureus EsxAB.
  • SEQ ID NO: 32 S. aureus StaO l 1 nucleotide sequence.
  • SEQ ID NO: 33 S. aureus Sta069 LysM3a 29-71.
  • SEQ ID NO: 38 S. aureus StaO l 1 polypeptide sequence.

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Abstract

L'invention concerne le domaine des adjuvants et des agonistes de TLR, en particulier des agonistes de TLR2, destinés à être utilisés en tant qu'adjuvants.
PCT/EP2015/058535 2014-04-21 2015-04-20 Adjuvants de vaccins WO2015162100A1 (fr)

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CN114539356A (zh) * 2022-02-21 2022-05-27 中国人民解放军陆军军医大学 一种脂肽及其制备方法和应用
CN114539356B (zh) * 2022-02-21 2023-06-09 中国人民解放军陆军军医大学 一种脂肽及其制备方法和应用

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